Available online www.jocpr.com

Journal of Chemical and Pharmaceutical Research, 2012, 4(5):2614-2625

Research Article ISSN : 0975-7384 CODEN(USA) : JCPRC5

Synthon Disconnection Strategy for the Synthesis Design of “Coelenterazine”- A Bioluminescent Marine Natural Product used in Bioassays

Chittaranjan Bhanja 1*, Subhendu Chakroborty 2

1Department of Chemistry, Utkal University, Bhubaneswar-751004, Odisha, India 2Department of Chemistry, Ravenshaw University, Cuttack-753003, Odisha, India ______

ABSTRACT

Synthon disconnection strategy is a problem solving technique in the planning of organic syntheses. This strategy, developed by Prof. E.J.Corey of Harvard University, plays vital role in the synthesis design of many bioactive natural products used in analytical biochemistry, molecular biology and drug discovery process. Keeping an overview on the published works in both journals and patent literatures, a good number of synthesis schemes have been proposed based on this strategy for a bioluminescent natural product ‘Coelenterazine’ isolated from marine organism jellyfish Aequorea victoria that finds extensive applications in bioassays. The proposed synthesis planning being a theoretical investigation, the actual laboratory execution requires the cross examination of a considerable number of factors such as reactions, reagents and order of events. In actual practice, generally that route is most feasible which is cost-effective, safe, and easy to carry out and gives maximum yield in a short reaction time.

Keywords: Anti-oxidant, Bioassays, Bioluminescence, Coelenterazine, Synthon disconnection strategy, . ______

INTRODUCTION

Organic synthesis is one of the major activities of organic chemists and the planning of synthesis is an intellectual task where chemist’s imagination, art, creativity and knowledge need to be explored. The simplest synthesis of a is one in which the target molecule can be obtained by submitting a readily available starting material to a single reaction that converts it to the desired target molecule. However, in most cases the synthesis is not straightforward; in order to convert a chosen starting material to the target molecule, numerous steps that add, change, or remove functional groups and steps that build up the carbon atom framework of the target molecule may need to be done. A systematic approach for designing synthetic routes to a molecule is promulgated as a result of Prof. E.J.Corey’s developments of ‘synthon disconnection approach’/‘retrosynthetic analysis’[1-3]. Retrosynthetic analysis is a protocol of stepwise breaking of target molecule to starting materials by disconnection of bonds and functional group interchange to a sequence of progressively simpler structures along a pathway just reverse of actual synthesis. Every disconnected part is an idealized fragment, called ‘synthon’. The when joined by known or conceivable synthetic operations result in the formation of target molecule (TM). Retrosynthetic analysis of a target molecule usually results in more than one possible synthetic route. It is therefore necessary to critically assess each derived route in order to choose the single route that is most feasible. In actual practice, generally that route is selected which is cost-effective, safe (the toxicity and reactivity hazards associated with the reactions involved), and

2614 Chittaranjan Bhanja et al J. Chem. Pharm. Res., 2012, 4(5):2614-2625 ______easy to carry out and produces maximum yield in a short reaction time, when assessing alternative synthetic routes to a molecule.

Organic compounds from marine organisms serve as compounds of interest both in their natural form and as templates for design and discovery of bioactive compounds that play important role in analytical biochemistry, molecular biology and drug discovery process. [4-9]. Bioluminescence is the production and emission of light by a living organism as the result of a chemical reaction during which chemical energy is converted to light energy. This phenomenon is exhibited by many marine organisms such as the sea pansy Renilla reniformis , the deep-sea shrimp Oplophorus gracilirostris , jellyfish Aequorea victoria , the squid Watasenia and hydroid Obelia [10-13]. and has been used extensively in different formats for life science research and drug discovery owing to its extremely high sensitivity and nonhazardous nature. The luminescent system of the jellyfish Aequorea victoria consists of the photoprotein aequorin, which contains the molecule “Coelenterazine” fig.1 as a prosthetic group and shows considerable potential in this area. The bioluminescence reaction of coelenterazine involves an oxidative process with molecular oxygen to produce the emission of light. In bioluminescent applications, coelenterazine has been used with the photoprotein aequorin and its recombinant analogues to monitor Ca 2+ concentrations in mammalian cells[14].Analogues of coelenterazine are also used with aequorin to study ion channels and tyrosine kinase receptors[15] Furthermore, fluorescence resonance energy transfer (FRET) assays that use coelenterazine to detect protein-protein interactions have been developed[16]. Aside from being a luminescent compound, Coelenterazine has also been studied for its anti-oxidant properties for protection against the oxygen toxicity in the environment [17]. Coelenterazine is distributed in a very small amount in its natural sources due to which the quantitative extraction and purification is a very difficult task. Therefore, synthetic methodologies for rapid access of this molecule and its templates are highly essential in synthetic organic chemistry/medicinal chemistry, for a better understanding of its chemiluminescent properties and applications in analytical biochemistry.

O OH N N

N H HO

Figure I: Coelenterazine

Although a few methods of synthesis of Coelenterazine are well documented in literature [18], some alternative synthetic routs are still required for its commercial success. Keeping an overview on the published works both in journals and patent literatures, an effort has been made to propose a good number of synthesis schemes for Coelenterazine based on synthon disconnection approach /retrosynthetic analysis. To the best of our knowledge, this type of work has not been reported earlier. The choice of this molecule for synthesis planning is obvious as bioassays are routinely works in life science research related to drug discovery. In this profit oriented world, the chemical/ pharmaceutical industries are also vibrant today in search of cost effective scalable synthesis. Moreover with the availability new reagents, chemical reactions, sophisticated new methods of laboratory execution and the application of synthon approach to analyse the target leading to several routes have made it possible to rethink their synthesis for the improvement in existing processes to satisfy the commercial need.

EXPERIMENTAL SECTION

The structure and information about Coelenterazine as bioluminescent candid has been collected from different books [1, 2]. The proposed synthesis plannings are then exploited in a novel way from the result of its retrosynthetic analysis using the basic principle outlined in the pioneering works of Prof. E.J. Corey. The symbols and abbreviations are synonymous to that represented in book [3].The analysis–synthesis schemes being theoretical propositions; obviously the syntheses have not been executed in the laboratory. The actual laboratory execution requires the cross examination of a considerable number of factors such as reagents, reactions, order of events, economical viability, environmental benign, saftyness, short time and scalable synthesis.

2615 Chittaranjan Bhanja et al J. Chem. Pharm. Res., 2012, 4(5):2614-2625 ______RESULTS AND DISCUSSION

Retrosynthetic Analysis: 1

O O OMe N OH N N N N NH C-N 2 FGI amide H OMe N N N H Protection H C=N + HO MeO imine O O TM 1 MeO 2 3

N NH2 N NH2 N NH2 Me N NH2 C-C BuSn3 FGI C-C N + Br N N + N MeO MeO 2 4 5 6 7 8

OEt S S H OMe FGI S OMe C-C O + Li OMe O O S O S S 3 9 10 11 12

Synthesis: 1

CH N NH N NH N NH2 3 2 2 MeO BuSn N NH2 3 n-BuLi Bu4NBr3,Py 4 + N N CHCl ,r.t. Br N N THF 3 Pd(PPh3)4 5 MeO 2 8 7 6 DMF

OEt O S n-BuLi S OMe HgCl ,CH CN Li 10 S OMe 2 3 H OMe S S Ca(CO3)2,H2O S O O O 12 11 9 3 O O N NH 2 OMe OH H OMe Con.HCl N N aq.HCl N N + N (TM) O O EtOH,reflux 1,4-dioxane 2 3 N N MeO 1 H H MeO HO

Scheme: 1

Regioselective alkylation of 2-amino pyrazin 8 with benzyllithium prepared from toluene 6 & n-butyl lithium in THF, forms 2-amino-3-benzylpyrazine 6. Selective bromination of 6 using tetra-n-butylammonium tribromide(Sato’s method)[19] produces 3-benzyl-5-bromo-2 aminopyrazine 5. Treatment of 5 with organostannane reagent 4 in presence of Pd(PPh 3)4 in DMF undergoes Stille coupling to form 2. p-methoxyphenylglyoxal 3 is prepared from ethyl p-methoxyphenyl acetate 10 by exploring dithiane based-route. Coupling of 2 with glyoxal 3 under acidic condition forms 1.Deprotection of methyl ether group of 1 under mild acidic condition forms the target molecule (TM ).

2616 Chittaranjan Bhanja et al J. Chem. Pharm. Res., 2012, 4(5):2614-2625 ______Retrosynthetic Analysis: 2

O O N NH OH OAc 2 OAc N N N N O C-N N H FGI amide + N N H protection H C=N HO O HO imide 14 HO TM 13 15

OH O N CN N NH N NH O N NH2 2 2 NH2 FGI FGI 2 x C-N N N N + protection HO MeO MeO OMe 14 2 16 17 18 O O H

19OMe 20 OAc OAc OAc OAc OH O O O O O H Br Cl HO HO 15 21 O 22 23 24

Synthesis: 2

O OH N N O CN O O O H NH2 .HCl 1.NaHSO3,H2O n-Bu-ON=O 0 2.28% NH4OH,55-60 C 3.NaCN 4.alcoholic HCl ' 18 19 OMe OMe 17 OMe 20 OH CN O N NH2 N N NH2 NH2 .HCl i)H2/Raney-Ni,EtOH TiCl4,Py N O + N ii)Py/HCl, HO MeO MeO 14 18 16 17 OH OAc OAc O O OAc NaOAc O i)CH N O SOCl2 2 2 i)AgNO ,CH CN Ac O 3 3 HO 2 HO Cl ii)HBr Br 24 23 22 21 ii)CH3COONa.3H2O, OAc DMSO O H 15 O

2617 Chittaranjan Bhanja et al J. Chem. Pharm. Res., 2012, 4(5):2614-2625 ______

O N NH2 OH OAc N O N EtOH/H O/HCl N 2 + H (TM) 15 N HO 14 O H HO

Scheme: 2 p-methoxyphenylglyoxal aldoxime 17 is formed from p-methoxyphenyl ethanone 19 on treatment with n-Butyl nitrite and subsequent tautomerisation.The α-amino nitrile 18 is prepared from 2-phenyl 20 by following Freifelder and Hasbrouck method[20]. Nucleophilic addition of 18 to the carbonyl group of 17 in presence of TiCl 4/Py forms pyrazine N-oxide which is reduced and deprotected to give aminopyrazine 14 .The phenyl acetic acid 23 prepared from 24 is then transformed to α-bromoketone 21 and then benzylglyoxal 15 .Cyclization of 14 with 15 in an acidic medium forms the target molecule ( TM ).

Retrosynthetic Analysis: 3

O OH N N N NH C-N 2 H OH amide + N N C=N O O H imine HO HO TM 14 25

N NH2 N NH2 N NH2 C-C B(OH) FGI O FGI O 2 N N N + protection MeO HO HO MeO 14 26 27 28

N Cl N Cl N NH2 N NH2 CHO FGA FGI FGI C-C N Cl O O O OH Br N N N N + N 29 30 31 32 33 34

OH FGI O OH O H OH Cl + CH2N2 Cl O O 35 37 25 36 Synthesis: 3

N Cl N Cl N NH N Cl Li TMP N NH2 2 NH Br2,K2CO3 THF MnO2 3 OH THF O EtOH, O AcOH,r.t. O N CHO N N N Br N 32 29 33 34 31 30

2618 Chittaranjan Bhanja et al J. Chem. Pharm. Res., 2012, 4(5):2614-2625 ______

N NH2 N NH N NH MeO B(OH)2 2 2 28 N H aq.KOH Cl Pd(dppb),aq.Na CO O EtSNa O 2 4, 2 2 3 N N N (CH OH) , DMF, 2 2 Ph Me,EtOH,reflux 26 high temp. MeO 27 HO HO 14

OH OH OH O O O DMSO, H OH CH2N2 N2 Cl Cl 37 Kornblum Oxdn O O Cl 35 25 36 N NH2 O OH N N N H OH Con.HCl + (TM) HO 14 O O 25 EtOH,reflux N H HO

Scheme: 3

Condensation of 2-chloropirazine 33 with benzaldehyde 34 in presence of a strong base 2,2,6,6-tetramethylpiperidyl lithium (LiTMP) forms the chloropyrazinoalcohal 32 .Oxidation of 32 by MnO 2 forms the ketopyrazine 31 .The chlorine substitution is then achieved using NH 3 in EtOH furnishing the aminopyrazine 30 .Bromination of aminopyrazine by Br 2/K 2CO 3 affords the bromopyrazine 29 .The bromopyrazine then undergoes Suzuki coupling with p-methoxyphenylboronic acid 28 to form the aminopyrazine 27.Deprotection of methyl aryl ether group of 27 using ethanethiolate in DMF to form 26 .Wolf-Kishner reduction of 26 affords the aminopyrazine 14 . Diazomethylation and subsequent decomposition of p-hydroxyphenyl acetyl chloride 36 forms corresponding α- chloro ketone 35.Kornblum oxidation of 35 affords p-hydroxybenzylglyoxal 25 Coupling of 14 with glyoxal 25 under acidic condition forms the target molecule ( TM ).

Retrosynthetic Analysis: 4 O O OH OBn N NH2 N N N N C-N EtO FGI amide N + OBn protection N N H C=N EtO H imine HO O HO TM HO 38 14 39

B(OH) N NH2 N NH2 CH N NH2 N NH2 2 3 FGI C-C FGI C-C N NH2 + Br N N + N protection N 40 N 14 2 28 5 8 7 HO MeO OMe

EtO Cl HO HO OBn C-C O OEt FGI FGI + OBn OBn OH EtO 43 44 O 39 EtO 41OEt 42

2619 Chittaranjan Bhanja et al J. Chem. Pharm. Res., 2012, 4(5):2614-2625 ______Synthesis: 4 CH3 N NH N NH2 N NH2 2 MeO B(OH)2 n-Bu Li Bu NBr ,Py + 4 3 28 N N CHCl ,r.t. Br N (PhCN)2,PdCl2 TMEDA,THF 40 3 5 (Ph) P(CH ) P(Ph) 8 7 2 2 2 2 1M aq.Na2CO3 tolune,ethanol

N NH2

N NH2 Py/HCl N N HO 14 2 MeO

Cl N Cl NN HO HO Cl EtO BnCl Mg,THF OH OBn Cl OBn OBn K CO 44 2 3 43 DMF,CH2Cl2 42 O OEt EtO EtO OEt O 39 41 O N NH2 OH EtO aq.HCl/1,4-dioxan N N OBn reflux N (TM) EtO O 39 N HO 14 H HO

Scheme: 4

3-benzyl-5-bromo-2 aminopyrazine 5 is produced from 2-aminopyrazine 8 as discussed in scheme :1. Treatment of 5 with commercially available p-methoxyphenylboronic acid 28 in presence of 1,4-bis (diphenylphosphino)butane and bis (benzonitrile)dichloropalladium II gives 3,5 disubstituted aminopyrzine 2.The methoxy group of 2 is cleaved using pyridine hydrochloride to afford the aminopyrazine 14 . Benzylprotected p-hydroxybenyl alcohol 43 obtained from p-hydroxybenzylalcohol 44 is chlorinated by using cyanuric chloride in DMF to form p-benzoyloxy benzyl chloride 42.The chloride is then converted in to its corresponig and then subjected to a reaction with the commercially available ethyl dietoxyacetate 41 to form highly protected dicarbonyl compound 39.Coupling of 14 with glyoxal 39 under acidic condition forms the target molecule (TM ).

Retrosynthetic Analysis: 5 O O OSMDBT OSMDBT OH N N N N N NH2 C-N FGI amide EtO + N protection N C=N N H H imine EtO O TBDMSO TBDMSO 46 47 HO TM 45

B(OH)2 MgCl N NH2 N NH2 N NH C-C 2 N NH2 + C-C N Br N + Br N Br N TBDMSO 46 48 OSMDBT 5 49 50 8

2620 Chittaranjan Bhanja et al J. Chem. Pharm. Res., 2012, 4(5):2614-2625 ______

OSMDBT OSMDBT OSMDBT OH C-C O OEt FGI FGI EtO + Cl HO HO OEt 47 EtO 41 51 52 44 EtO O

Synthesis: 5 N NH N NH2 2 N NH2 TBDMSO B(OH) N NH2 MgCl,ZnCl2 2 NBS 49 48 N DCM,r.t. (PhCH2)2PdCl2 N Br N Br (Ph3P)2PdCl2,r.t. Br N (Ph)2P(CH2)4P(Ph)2 8 5 TBDMSO 50 ethanol,aq.Na2CO3 46 tolune,reflux

OSMDBT OH OSMDBT OSMDBT Mg,THF 4-DMAP,Imidazole MSCl,Et N HO 3 Cl TBDMSCl,CH Cl HO O OEt EtO 2 2 CH2Cl2 44 52 51 EtO OEt 47 41 EtO O OSMDBT O O N NH2 OSMDBT OH N N N N N EtO aq.HCl/1,4-dioxan + (TM) 47 reflux TBDMSO EtO O N N 46 H H TBDMSO 45 HO

Scheme: 5

Bromination of 2-aminopyrazine 8 with N-bromosuccinamide in DCM forms 3,5-dibromo-2-aminopyrazine 50. Palladium catalysed coupling of 50 with benzylzinc chloride reagent 49 (formed from benzylmagnesium chloride and anhydrous ZnCl 2) affords 3-benzyl-5-bromo-2 aminopyrazine 5. Under Suzuki coupling condition, 5 condenses with boronic acid 48 to afford 46. Acid catalysed addition of α-keto acetal 47 (formed as above) to 46 forms 3,7- dihydroimidazo[1,2a]pyrazine-3-one 45.Deprotection of silyl groups of 45 under mild acidic condition affords the target molecule ( TM ).

Retrosynthetic Analysis: 6 O O OH OMe N N N N N NH2 O C-N FGI amide N N + O N protection H C=N H imine O OMe HO TBDMSO TBDMSO 54 TM 53 46

Br Br N NH2 N NH2 N NH2 C-C C-C FGA N NH2 + + N Br N Br N Br N TBDMSO 46 55 OSMDBT 5 56 50 8

O O CHO H O protection O S S S S S S O 54 OMe MeO MeO 58MeO 59 57

2621 Chittaranjan Bhanja et al J. Chem. Pharm. Res., 2012, 4(5):2614-2625 ______

protection H O MeO 60

Synthesis: 6

Br N NH2 N NH2 56 N NH2 TBDMSO Br N NH2 NBS 0 55 Zn,I2,5%, DMA,80 C 1.n-butyllithium,ZnCl2/THF N DCM/RT Br N Br Cl Pd(PPh ) ,r.t. Br N N 2 3 2 2.Cl2Pd(PPh3)2,r.t. TBDMSO 46 8 50 5

0 H H 1.n-BuLi ,-20 C CHO OH SH SH , p-TsOH S S S S OH O BF ,OEt 2.DMF PhMe,reflux MeO 60 3 2 MeO 59 MeO 58 DCM O O

O NCS,AgNO3 O S S MeCN/H O O 54 MeO 2 OMe 57 O N NH2 OH O N N Con.HCl,EtOH,reflux N + O (TM) N H TBDMSO 46 O 54 OMe HO

Scheme: 6

Bromination of 2-aminopyrazine 8 with NBS forms 3,5-dibromo-2-aminopyrazine 50 .Negishi cross-coupling reaction of 50 with benzyl zincbromide formed from benzyl bromide 56 by Huo’s method [21] to form 3-benzyl-5- bromo-2 aminopyrazine 5. The in situ generated phenyl lithium organometallic from tetra -butyldimethylsilyl (TBDMS) protected bromophenol 55 with n-butyl lithium is then treated with anhydrous ZnCl 2 to create the phenyl zinc chloride (Buchwald’s and Miline’s approach to zincation)[22] which undergoes cross coupling with the aryl bromide 5 using Pd (II) catalyst to form 46 . The aldehyde group of phenylacetaldehyde 60 is protected by dithiane and then lithiated by n-BuLi.The lithiated derivative on treatment with DMF forms another aldehyde 58. This aldehyde is then protected to the corresponding acetal to form thioketal acetal moiety 57.Selective hydrolysis of the thioketal acetal by N-chlorosuccinamide(NCS) and AgNO 3 /MeCN provides α-keto acetal 54.Condensation of 54 with 46 in acidic condition affords the target molecule ( TM ).

Retrosynthetic Analysis: 7 O O OH OAc N N N NH2 N N C-N FGI amide OAc N + O N protection N C=N H H imine HO H O HO HO 14 TM 61 15

2622 Chittaranjan Bhanja et al J. Chem. Pharm. Res., 2012, 4(5):2614-2625 ______

N Cl N NH N NH2 N NH2 2 FGA FGIO FGI O O N N N N protection MeO MeO 62 HO 14 HO 26 27

COCl N Cl C-C Cl N Cl C-C + N + MeO Cl N MeO 64 65 66 63 OAc OAc OAc OAc O O O Cl H 15 67Br 21 68 O ONO2

Synthesis: 7 ZnCl 2 i)TMPMgCl.LiCl N Cl N Cl 65 N Cl 0 MeO THF,-45 C NH ,BuOH O 3 Pd(dba) (2 mol%) 2.ZnCl N 0 Cl N 2 N 2 180 C tfp(4mol%),THF,250C 3.PhCOCl 63 66 Pd(PPh ) MeO MeO 64 3 4 62 (Negishi cross-coupling) N NH N NH2 N NH2 2 N H aq.KOH O EtSH,NaH 2 4, O 0 N N DMF,1000C N (CH2OH)2,240 C 14 MeO 27 HO 26 HO

OAc OAc OAc OAc 0 O 1.Zn.LiCl,THF,25 C AgNO3.MgCN O Cl NaOAc.3H2O 2.CuCN,2LiCl 0 0 68 25 C DMSO,25 C 3.bromoacetyl chloride Br 21 ONO 67 O 15 2 O H O N NH2 OAc OH N N N + EtOH,HCl 14 O 15 (TM) HO reflux N O H H HO

Scheme: 7

Negishi cross-coupling of 2, 5-dichloropyrazine 66 with organozinc compound 65 forms the aryl pyrazine 64.Magnesiation of 64 with TMPMgCl.LiCl gives after transmetalation with ZnCl 2 and acylation with benzoyl chloride 63, the ketopyrazine 62 .Aminopyrazine 27 is then formed from 62 on treatment with NH 3in BuOH.Cleavage of the methyl ether with sod.thioethanoate in DMF yields the corresponding alcohol 26.Wolf- Kishner reduction of alcohol affords the coelenteramine 14 . Addition of Zn-dust to 4-(chloromethyl) phenyl acetate 68 and subsequent trapping with bromoacetyl chloride after transmetalation with CuCN.2LiCl, furnish the acetyl derivative 21.Nitration of 21 with AgNO 3 forms the nitrate ester 67 which upon reaction with NaOAc in DMSO

2623 Chittaranjan Bhanja et al J. Chem. Pharm. Res., 2012, 4(5):2614-2625 ______affords the corresponding acetoxy α- keto aldehyde 15 .Condensation of 14 with α- keto aldehyde 15 in 1:1 HCl/EtOH provides the target molecule ( TM) .

CONCLUSION

The planning of a multistep synthesis via synthon disconnection approach/ retrosynthetic analysis is a challenging task that requires not only a thorough knowledge of synthetic reactions, but also a logical approach for disconnecting a complex target molecule into simpler or commercially available starting materials for a chemical synthesis. It is a paper exercise; a full analysis of this type will provide many routes for synthesizing the target molecule. Taking the privilege of this approach, we have proposed a good number of synthesis schemes for bioluminescent natural product “Coelenterazine”. Being a theoretical proposition, the synthesis works have not been executed in the laboratory. Strategic application of this approach can determine different routes to synthesize the target molecule even if the target molecule has never been synthesized earlier. Scalable synthetic routes for newly discovered natural products, drug molecules and useful compounds not available in adequate quantities from natural resources can be best provide by this approach. With the advancement and development of new reactions and reagents, the synthesis of best selling drugs can be rethinking for the improvements in existing process through this approach.

Acknowledgements The author CB deeply acknowledges UGC, New Delhi, India for providing financial support as Teacher Fellow grant & author SC deeply acknowledges CSIR, New Delhi, India, for providing financial support as SRF. Both authors also thank the authorities of IIT Bhubaneswar, IMMT Bhubaneswar and NISER, Bhubaneswar for permission to collect information from books and journals from their library.

REFERENCES

[1] E J Corey. Pure. Appl. Chem ., 1967, 14 , 19. [2] E J Corey; WT Wipke. Science., 1969, 66 ,178. [3] E J Corey. Q. Rev .Chem. Soc ., 1971, 25 ,455. [4] M Chalfie; Y Tu; G Euskirchen ; WW Ward; DC Prasher. Science., 1994 ; 263,802 -805. [5] WW Ward; GC Swiatek; DG Gonzalez. Methods Enzymol ., 2000 ; 305, 672-680. [6] C H Contag , M H Bachmann. Annu. Rev. Biomed. Eng ., 2002 , 4, 235-260. [7] L M DiPilato , X Cheng , Zhan. J.Proc. Natl Acad. Sci . USA., 2004 ; 101 :16513-16518. [8] M J Hayes; C J Merrifield ; D Shao ; J AyalaSanmartin; C D Schorey ; T P Levine ; J Proust ; J Curran ; M Bailly ; SE Moss. J. Biol. Chem ., 2004 ; 279, 14157 -14164. [9] J E Gonzalez; P A Negulescu.Curr. Opin. Biotechnol., 1998 , 9,624 -631. [10] CM Thomson; P J Herring; AK Campbell. J. Biolumin. Chemilumin., 1997 , 8 , 87-91. [11] K Jones; F Hibbert,; M Keenan. Trends Biotechnol., 1999 , 17 , 477-481. [12] K Hori; MJ Cormier. Proc. Natl. Acad. Sci. U.S.A., 1973 , 14 , 120-123. [13] JW Hastings; CH Johnson. Methods Enzymol., 2000 , 305 , 75-104. [14] V Robert ; P Pinto; V Tosello; R Rizzuto,; T Pozzan. Methods Enzymol., 2000 , 327 , 440-456. [15] VJ Dupriez; K Maes; E Le Poul; E Burgeon; M Detheux. Receptors and Channels., 2002 , 8, 319-330. [16] Y Xu ; A Kanauchi; A.G Von Arnim; DW Piston; CH Johnson. Methods Enzymol., 2003 , 36 , 289-301. [17] ML Dubuisson; B de Wergifosse; A Trouet; F Bague ; J Marchand-Brynaert ; JF Rees. Biochem Pharmaco l., 2000 , 60 (4), 471-478. [18] a) Y .Kishi; H Tanino; T Goto. Tetrahedron Letter.,1972 ,13 ,2747-2748.b) S Inoue; S Suguira; H Kakoi; K Hasizume. Chem. Lett., 1975, 4, 141-144.c) K Jones; M Keenan; F Hibbert. Synlett., 1996 , 509-510.d) G Gonzalez- Trueba; C Paradisi; M Zoratti. Anal.Biochem .1996 ,240 ,308-310.e) M Keenan; K Jones; F Hibbert. Chem.Commun ., 1997 ,323-324.f) K Jones; F Hibbert; M Keenan. Trends Biotechnol., 1999 , 17 , 477-481.g) M Adamczyk ; SR Akireddy; DD Johnson; Mattingly,PG ; Y Pan,; RE Reddy. Org. Prep. Proced .Int., 2001 , 33 (5) ,477-485. h) M Adamczyk; SR Akireddy; DD Johnson; PG Mattingly;Y Pan; RE Reddy. Tetrahedron., 2003 , 59 , 8129-8142. i) M Mosrin; T Bresser; P Knochel. Org.Lett., 2009 , 11 (15), 3406-3409. [19] N Sato. J.Heterocyclic Chem., 1972, 19,663 . [20] M Freifelder; R Hasbrouck. J.Am.Chem.Soc., 1960,82,696-698. [21] S Hou. Org.Lett.,2003 ,5,423-425. [22] J E Milne; S L Buchwald. J. Am. Chem. Soc. , 2004 , 126 (40), pp 1302–1303.

2624 Chittaranjan Bhanja et al J. Chem. Pharm. Res., 2012, 4(5):2614-2625 ______[1] RW Sabnis.“Handbook of Biological Dyes and Stains: Synthesis and Industrial Applications” John Wiley & Sons, 2010 . [2] A Roda. Chemiluminescence and Bioluminescence: Past, Present and Future RSC Publishing, 2010 . [3] E J Corey; X M Chang. “The Logic of Chemical Synthesis” Wiley, New York, 1989. [4] S Warren. “Designing Organic Synthesis: A Programme Introduction to Synthon Approach”, John Wiley & Sons,New York, 1978. [5] S Warren. “Organic Synthesis-The Disconnection Approach”. John Wiley & Sons, 1982 . [6] JH Fuhrhop; G Li. “Organic Synthesis: Concepts and methods” WILEY-VC GmbH & Co.KGaA 2003 . [7] J Clayden, N Greeves, S Warren, P Wothers. “Retrosynthetic analysis, In Organic Chemistry” Oxford University Press Inc., New York, 2001 ; pp. 773-778. [8] RK Kar. “Fundamentals of Organic Synthesis: The Retrosynthetic Analysis”. NCBA, Kolkata, India. 2007. [9] JJ Li; GW Gribble. Tetrahedron Organic Chemistry Series, Vol-26 “Palladium in Heterocyclic Chemistry: A Guide for the Synthetic Chemist (2/e) Gulf Professional Publishing, 2007 [10] JF Case. “Bioluminescence and Chemiluminescence “World Scientific Publishing Co.Pte.Ltd.P.O.Box 128 , Farrer Road , Singapore 912805, 2001. [11] O Shimomura.“Bioluminescence: Chemical Principles and Methods ” World Scientific Publishing Co.Pte.Ltd. 5 Toh Tuck Link, Singapore 596224, 2006.

2625